Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Virginia, 2007. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Le Fas Ligand (FasL) est le déclencheur naturel de l’apoptose induite par le récepteur Fas. Il est produit sous la forme d’une protéine membranaire homotrimérique, qui peut être clivée par une métalloprotéase pour engendrer une forme soluble (sFasL). Cette forme conserve sa structure homotrimérique, mais son activité pro-apoptotique est très faible car elle n’atteint pas un degré de polymérisation suffisant. Au laboratoire, une molécule sFasL hautement polymérique a été obtenue en fusionnant un domaine de type immunoglobuline au domaine extracellulaire du FasL. Ce domaine permet de polymériser le sFasL sous forme hexamérique et dodécamérique. Cette molécule appelée pFasL possède une activité anti-tumorale in vitro et in vivo dans un modèle de greffe de cellules humaines tumorales à des souris immunodéficientes. Dans cette étude, nous nous sommes focalisés sur l'amélioration de pFasL, avec deux objectifs complémentaires. Tout d'abord, nous avons fusionné un récepteur à l’antigène de lymphocytes Tγδ (TCRγδ) au pFasL (TCR-pFasL) de façon à orienter son activité vers les cellules tumorales carcinomateuses exprimant l’antigène spécifique de ce TCR, qui est l’EPCR (Endothelial Protein C Receptor). Ensuite, nous souhaitions améliorer la production et l’activité spécifique de pFasL et son dérivé TCR-pFasL. La stratégie de ciblage dépendante du TCR n’a pas été validée dans cette étude, mais nous avons pu décrire une approche originale pour améliorer la production et/ou l’activité cytotoxique de ces chimères en co-exprimant celles-ci avec du sFasL non apoptotique. Cette approche a été validée avec deux autres chimères obtenues à partir de pFasL, l’une contenant la molécule de présentation antigénique HLA-A2 et la seconde le ligand CD80 du récepteur CD28, pour lequel le ciblage fonctionne. Notre étude ouvre ainsi des perspectives pour appliquer ce protocole à une grande variété de protéines de fusion dérivées du sFasL pour des applications diverses, en recherche et en thérapie. / Fas ligand (FasL) is the natural trigger for apoptosis induced by the Fas receptor. FasL exists as an homotrimer on the cell surface, which can be cleaved by a metalloprotease to generate a soluble form (sFasL). The sFasL form retains its homotrimeric structure, but displays almost no pro-apoptotic activity because it does not reach a sufficient degree of polymerisation. In the laboratory, a highly polymeric molecule sFasL was obtained by fusing an immunoglobulin-like domain to the extracellular domain of FasL. This domain polymerises the molecule through disulphide bonds, leading to hexameric and dodecameric compounds. This molecule, called pFasL, possesses an anti-tumor activity in vitro but also in vivo in a model of human tumor cell transplanted to immunodeficient mice. In this study, we focused on improving pFasL with two complementary objectives. Firstly, we fused to pFasL an antigen receptor of γδ T-cells (TCRγδ, leading to TCR-pFasL) to direct its activity towards carcinoma cells expressing this TCR specific antigen, which is the EPCR (Endothelial Protein C Receptor). Then, we wanted to improve the production and specific activity of pFasL and its derivative TCR-pFasL. In this study, the TCR dependent targeting strategy has not been validated, but we have described a novel approach to improve the production and/or cytotoxic activity of these chimeras by coexpressing them with non-apoptotic sFasL. This strategy has been validated with two other chimeras obtained from pFasL, one containing the antigen-presenting molecule HLA-A2 and the second one CD80, the ligand for the CD28 receptor. For this latter one, the cell targeting activity proved efficient. Our study opens up opportunities to improve and develop a wide variety of sFasL-derived fusion proteins for various applications in research and therapy.

Kit Yu Fu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 99-106). / Chapter I. --- Abstract --- p.vii / Chapter II. --- Acknowledgements --- p.viii / Chapter III. --- List of Figures --- p.ix / Chapter IV. --- Chapter --- p.1 / Introduction --- p.1 / Chapter 1.1 --- Laboratory diagnosis of infectious diseases --- p.1 / Chapter a. --- Culture --- p.1 / Chapter b. --- Direct detection by visualization --- p.2 / Chapter c. --- Direct detection by DNA or RNA hybridization --- p.2 / Chapter d. --- Detection by immunological methods (antigen or antibody detection) --- p.3 / Chapter 1.2 --- Types of antigen preparations / Chapter a. --- Crude antigenic extracts --- p.5 / Chapter b. --- Affinity-purified antigens --- p.6 / Chapter c. --- Recombinant antigens --- p.6 / Chapter 1.3 --- Methods of gene transfer to mammalian cells --- p.8 / Chapter 1.4 --- The immunoglobulins --- p.10 / Chapter 1.4.1 --- Ig structure --- p.11 / Chapter 1.4.2 --- Ig genes --- p.13 / Chapter 1.4.3 --- Ig gene rearrangement --- p.15 / Chapter 1.4.4 --- Recombinant Ig --- p.15 / Chapter 1.4.5 --- Myeloma-derived recombinant Ig (chimeric antibodies) --- p.17 / Chapter 1.4.6 --- Ig expression vectors --- p.19 / Chapter 1.5 --- Trichinella spiralis and trichinosis --- p.20 / Chapter 1.5.1 --- The parasite --- p.21 / Chapter 1.5.2 --- Antigens of T. spiralis --- p.21 / Chapter 1.6 --- Aim of present study --- p.25 / Chapter V. --- Chapter2 / Materials and Methods / Chapter 2.1 --- Chemicals --- p.27 / Chapter 2.2 --- Parasite --- p.27 / Chapter 2.3 --- Cell line and expression vectors --- p.28 / Chapter 2.4 --- Extraction of total RNA from T. spiralis --- p.29 / Chapter 2.5 --- Preparation of cDNA fragment from T. spiralis --- p.29 / Chapter 2.6 --- Characterization of Trichinella cDNA fragment --- p.31 / Chapter 2.6.1 --- By gel electrophoresis --- p.31 / Chapter 2.6.2 --- By restriction enzyme digestion --- p.31 / Chapter 2.7 --- Cloning of Trichinella cDNA fragment to g4R --- p.31 / Chapter 2.7.1 --- Preparation Trichinella cDNA fragment for ligation --- p.32 / Chapter 2.7.2 --- Preparation of g4R vector --- p.32 / Chapter 2.7.3 --- Ligation --- p.32 / Chapter 2.7.4 --- Transformation of Escherichia coli (TG1) / Chapter a. --- Preparation of competent cells for transformation --- p.33 / Chapter b. --- Transformation of competent cells by heat shock --- p.34 / Chapter 2.7.5 --- Screening of recombinant clones --- p.34 / Chapter 2.8 --- Preparation of fusion gene for transfection --- p.36 / Chapter 2.9 --- Introduction of DNA to myeloma cells by electroporation --- p.36 / Chapter 2.10 --- Enzyme-linked immunosorbent assay (ELISA) to detect fusion gene product / Chapter 2.10.1 --- Sandwich ELISA --- p.37 / Chapter 2.10.2 --- Detection of Trichinella antigen in fusion gene product --- p.38 / Chapter 2.10.3 --- Detection of Ig CH2-CH3 domains in fusion gene product --- p.38 / Chapter 2.11 --- Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.39 / Chapter 2.12 --- Genomic and transcriptional analysis of transfectants --- p.40 / Chapter 2.12.1 --- Genomic analysis of transfectants / Chapter a. --- DNA isolation --- p.40 / Chapter b. --- PCR amplification of the fusion gene fragment --- p.41 / Chapter 2.12.2 --- Isolation of fusion gene cDNA from transfectants --- p.41 / Chapter 2.12.3 --- Cloning of fusion gene cDNA to M13 mpl9 --- p.43 / Chapter 2.12.4 --- Preparation of single-stranded templates from M13 phage --- p.43 / Chapter 2.12.5 --- Dideoxy sequencing method (Sanger) --- p.44 / Chapter 2.12.6 --- Gel analysis of sequencing products --- p.44 / Chapter 2.13 --- Modification of the g4R expression vector by deletion of the CH2-CH3 exons 3' to the XhoI site / Chapter a. --- Partial EcoRI digestion of g4R --- p.45 / Chapter b. --- Addition of adaptors to the partial Eco RI-digested g4R vector --- p.46 / Chapter c. --- Preparation of modified g4R vector --- p.46 / Chapter 2.14 --- Cloning of the Trichinella P53 gene into the modified g4R vector --- p.46 / Chapter 2.15 --- Detection of Trichinella antigen in the second fusion gene product / Chapter a. --- Preparation of biotinylated mouse anti- Trichinella serum --- p.47 / Chapter b. --- Assay for the activity of biotin-Ts serum --- p.48 / Chapter c. --- "Assay for detection of Trichinella antigen in the fusion gene product from Tc2, Te1 and g4R transfected clones" --- p.48 / Chapter 2.16 --- Northern blot analysis of the RNA of transfected clones --- p.48 / Chapter a. --- RNA gel electrophoresis --- p.49 / Chapter b. --- RNA transfer --- p.49 / Chapter c. --- RNA hybridization --- p.49 / Chapter VI. --- "Chapter3 Construction of Ig-Trichinella fusion gene, Te1" / Chapter 3.1 --- Rationale of the gene construction --- p.51 / Chapter 3.2 --- Isolation of T. spiralis P49 gene by cDNA amplification --- p.55 / Chapter 3.3 --- Cloning of Trichinella P49 cDNA to g4R --- p.58 / Chapter 3.4 --- Screening of recombinant clones --- p.58 / Chapter VII. --- Chapter4 Characterization of Tc1 fusion gene product / Chapter 4.1 --- Transfection of fusion gene to J558L myeloma cells / Chapter 4.2 --- Antigenicity of fusion gene product with respect to Trichinella activity --- p.64 / Chapter 4.3 --- Detection of Ig CH2-CH3 domains in fusion gene product --- p.65 / Chapter 4.4 --- Size determination of fusion gene product --- p.69 / Chapter 4.5 --- Transcriptional and genomic analysis of transfectants producing the fusion gene product --- p.71 / Chapter 4.5.1 --- Genomic analysis --- p.71 / Chapter 4.5.2 --- Sequence analysis of transcript from the CH1 to CH2 exon --- p.71 / Chapter 4.5.3 --- Sequence analysis of transcript from the CH1 to CH3 exon --- p.77 / Chapter VIII. --- "Chapter5 Construction and characterization of second fusion gene, Tc2, using modified g4R vector" --- p.81 / Chapter IX. --- Chapter6 General Discussion / Use of recombinant DNA technology to produceantigen for use in the diagnosis of infectious diseases --- p.89 / Characterization of the fusion gene product --- p.89 / Absence of Trichinella sequence in fusion gene product due to exon skipping --- p.94 / A new strategy for producing Ig fusion proteins: modification of the g4R vector --- p.96 / Prospect of utilizing Ig expression system for producing antigen --- p.97 / Chapter X. --- References --- p.99 / Chapter XI. --- Appendix --- p.107

by Kar-Keung Ching. / "May 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 154-173). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Dosage compensation is the key regulatory process employed in <i>Drosophila melanogaster</i> to equalize the level of gene transcripts between the single X chromosome in males (XY) and the two X chromosomes in females (XX). Dimorphic sex chromosomes evolved by the severe degeneration of the Y chromosome, giving rise to an imbalance between the heterogametic sex and the homogametic sex. Vital to the viability of male Drosophila is the dosage compensation complex (DCC), a ribonucleoprotein complex that mediates the precise two-fold transcription of the single male X chromosome. The DCC is comprised of five proteins: male-specific-lethal proteins (MSL) 1, 2, and 3, male absent-on-the-first (MOF), maleless (MLE), and two non-coding RNAs. The complex specifically co-localizes along the male X chromosome in a reproducible manner, resulting in acetylation of lysine 16 of the N-terminal tail of histone H4. The exact mechanism of recruitment and spreading of the DCC along the male X chromosome remains unclear; recent studies propose a multi-step mechanism involving DNA sequence elements, epigenetic marks, and transcription. Understanding how dosage compensation functions provides insight into the interplay between gene regulation and chromatin remodelling. The goal of this project was to better understand how <i>Drosophila</i> MSL1, MSL3, and MOF interact and how their interaction modulates MOFs acetyltransferase activity. Recombinant protein constructs were cloned and over-expressed in a bacterial expression system permitting future structure determination by X-ray crystallography. The dMSL1820-1039 construct consisted of the C-terminal domain, reported to be able to interact with both dMSL3 and dMOF. dMSL3186-512 contained the domain required for the interaction with dMSL1 and dMOF. dMOF371-827 was comprised of the catalytic domain, the CCHC zinc finger, and the chromodomain, as the N-terminal region does not encode any known domains. All three recombinant proteins were successfully cloned, over-expressed, and purified to homogeneity. Recombinant dMOF371-827 was determined to acetylate histones. Interaction studies using GST pull-down assays and size exclusion chromatography determined that dMSL1820-1039 and dMOF371-827 did not interact above background levels. Moreover, size exclusion chromatography revealed dMSL3186-512 and dMOF371-827 did not interact nor did the three recombinant proteins form a stable complex.

Dosage compensation is the key regulatory process employed in <i>Drosophila melanogaster</i> to equalize the level of gene transcripts between the single X chromosome in males (XY) and the two X chromosomes in females (XX). Dimorphic sex chromosomes evolved by the severe degeneration of the Y chromosome, giving rise to an imbalance between the heterogametic sex and the homogametic sex. Vital to the viability of male Drosophila is the dosage compensation complex (DCC), a ribonucleoprotein complex that mediates the precise two-fold transcription of the single male X chromosome. The DCC is comprised of five proteins: male-specific-lethal proteins (MSL) 1, 2, and 3, male absent-on-the-first (MOF), maleless (MLE), and two non-coding RNAs. The complex specifically co-localizes along the male X chromosome in a reproducible manner, resulting in acetylation of lysine 16 of the N-terminal tail of histone H4. The exact mechanism of recruitment and spreading of the DCC along the male X chromosome remains unclear; recent studies propose a multi-step mechanism involving DNA sequence elements, epigenetic marks, and transcription. Understanding how dosage compensation functions provides insight into the interplay between gene regulation and chromatin remodelling. The goal of this project was to better understand how <i>Drosophila</i> MSL1, MSL3, and MOF interact and how their interaction modulates MOFs acetyltransferase activity. Recombinant protein constructs were cloned and over-expressed in a bacterial expression system permitting future structure determination by X-ray crystallography. The dMSL1820-1039 construct consisted of the C-terminal domain, reported to be able to interact with both dMSL3 and dMOF. dMSL3186-512 contained the domain required for the interaction with dMSL1 and dMOF. dMOF371-827 was comprised of the catalytic domain, the CCHC zinc finger, and the chromodomain, as the N-terminal region does not encode any known domains. All three recombinant proteins were successfully cloned, over-expressed, and purified to homogeneity. Recombinant dMOF371-827 was determined to acetylate histones. Interaction studies using GST pull-down assays and size exclusion chromatography determined that dMSL1820-1039 and dMOF371-827 did not interact above background levels. Moreover, size exclusion chromatography revealed dMSL3186-512 and dMOF371-827 did not interact nor did the three recombinant proteins form a stable complex.

The nuclear envelope is a highly organised double membrane system that separates the activities of the nuclear and cytoplasmic compartments in eukaryotic systems. The wide range of functions recently associated with the NE and the identification of hundreds of proteins associated with this cellular structure indicates that it is a major signalling node for the cell. Recent work indicates NE functions in signalling innate immune responses to herpesviruses. The viruses, on the other hand, often target or usurp NE functions in different ways. The NE is also a physical barrier that must be overcome for viruses like the herpesviridae that assemble capsids in the nucleus. This thesis addresses two important questions: 1) How do herpesviruses cross the NE after new viral particles are produced in the nucleus? and 2) What is the nuclear envelope role of NET23/STING in the activation of immune factors upon herpesvirus infection? To address the first question, I followed two different approaches. The first used the isolation of microsomes from HSV-1 infected cells to identify possible host factors involved during herpesvirus exit through the NE on the prediction that such proteins would disperse into the ER during infection. I identified a group of vesicle fusion proteins that play a role in this herpesvirus exit through the NE. Depletion of three identified vesicle fusion proteins decreased the growth of HSV-1 in host cells, yielding accumulation of viral particles in the nucleus. The second approach was to follow the fate of nuclear envelope transmembrane proteins (NETs) during HSV-1 infection. To address the question of how NET23/STING is involved in innate immunity I tested the hypothesis that this NET acts as a transport receptor to carry signals through the peripheral channels of the NPC when central channel transport is blocked by pathogens. FRAP was used to quantify the mobility of NET23/STING upon the induction of the innate immune response, finding an increase of the mobility for this protein in the NE. To further elucidate its role within the NE I tested whether some NE-NET23/STING binding partners were being redistributed between the nucleus and cytoplasm during innate immune responses. This revealed two of these binding partners normally redistribute upon innate immune response activation and this is blocked in cells knocked down for NET23/STING. Finally, I confirmed that NET23/STING contributes to chromatin remodelling during infection involving an increase in the H3K9Me3 epigenetic mark. Collectively, these data argue the identification of novel host proteins involved in herpesvirus nuclear egress and the finding of a new role for NET23/STING within the NE.

Gene therapy is a technique used to inactivate, replace or insert a corrective copy of a defective gene in order to help diseased tissues to function properly. Gene therapy is a promising treatment for many diseases cancer, cystic fibrosis, and Parkinson’s. There are different methods to introduce a gene to the cell; one of them is the use of viruses. Among viruses, lentiviruses have been popular vectors for gene delivery due to their efficient mode of gene delivery. However, the non-specific delivery of genes associated with viruses may result in undesirable side effects. Here, we propose a heterogeneous nanoparticle delivery system for targeted delivery of lentiviral particles containing a therapeutic gene. The heterogeneous nanoparticles (NPs) consist of the low density lipoprotein receptor 3 (LDLR3) and the keratinocyte growth factor (KGF), each fused to elastin-like-polypeptides (ELPs), LDLR3-ELP and KGF-ELP, respectively. Our results show that while homogeneous nanoparticles comprising of LDLR3-ELP alone blocked viral transduction, heterogeneous nanoparticles comprising of KGF-ELP and LDLR3-ELP enhanced viral transduction in cells expressing high levels of the KGF receptors (KGFR) compared to cells expressing low levels of KGF receptors. Overall, this novel design may help with the targeting of specific cells that overexpressed growth factor such as KGFR.